Project Summary As a chronic neuropsychiatric disease, addiction is associated with specific molecular and functional neuronal plasticity changes that are triggered by repeated drug exposure leading to persistent changes in neuronal function and ultimately behavior. One powerful mechanism that may underlie aspects of this persistence is epigenetics. Epigenetics (i.e. modulation of gene expression that occurs through altered chromatin structure without fundamental changes to the DNA sequence itself) has been shown to establish stable changes in cell function. These stable changes in cell function can give rise to remarkable changes at many levels of observation (e.g. neuronal plasticity, behavior). Currently, we still know very little about the epigenetic mechanisms that could establish the persistence characteristic of drug-seeking behavior and whether such mechanisms may also be involved in reinstatement, or other relapse-like behaviors. This proposal is focused on examining the molecular and cellular mechanisms that may be involved in reinstatement. More specifically, we will focus on the role of the medial habenula (MHb) in cocaine-induced reinstatement of drug-seeking behavior. Most studies investigating the MHb have focused on nicotine seeking due to the high concentration of nicotinic acetylcholine receptors found throughout the medial habenula-interpeduncular nucleus pathway. Recent studies have begun to implicate the MHb in cocaine- associated behaviors, yet the role of the MHb in regulating reinstatement of cocaine-seeking behavior remains largely unknown. In fact, the MHb is rarely included in reward circuitry diagrams. Our recent findings demonstrate that the MHb is engaged by cocaine-primed reinstatement and the activity of choline acetyltransferase (ChAT) expressing neurons in the MHb is sufficient to drive reinstatement (Lopez et al., 2018). These results suggest that the MHb is a powerful regulator of relapse-like behaviors, which has important implications for understanding the reward pathways in the brain related to relapse. We will also examine the role of a histone deacetylase, called HDAC3, and a key HDAC3 target gene, called Nr4a2, in MHb-dependent reinstatement of drug-seeking. HDAC3 is a key negative regulator of memory formation and associative plasticity, which functions by repressing the expression of Nr4a2. NR4A2 is a transcription factor that regulates aspects of dopamine signaling during development. Both HDAC3 and NR4A2 are highly expressed in the MHb within ChAT expressing neurons, indicating these important regulators of memory processes have a central role in behaviors associated with MHb-dependent reinstatement. In this proposal, we will test the central hypothesis that the MHb is a key regulator of reinstatement of cocaine- seeking behavior, and does so in an HDAC3/NR4A2-dependent manner. Successful completion of these studies will demonstrate the key nature of the MHb in reinstatement, identify the physiological processes in the MHb responding to cocaine, and identify key epigenetic regulators of MHb function.